The invention discloses an innovative method and apparatus by which the motion of charged particles in a solution subject to an applied electric field may be measured. Although the present invention will refer to macromolecules throughout much of its specification, the invention includes more generally all classes of small particles including emulsions, viruses, nanoparticles, liposomes, macro-ions and any other solution constituents whose size may lie between a half and a few thousand nanometers. Thus whenever the terms “molecule,” “macromolecule,” or “macro-ion” are used, it should be understood they include all of the aforementioned solution-borne objects.
Electrophoretic mobility is widely accepted as a sensitive probe of interfacial charge, and it has found numerous applications in both biological and colloidal samples. The correlation between electrophoretic mobility and colloidal stability, formulation stability and inter-molecular interactions has been and remains a subject of active research.
Electrophoresis is the migration of macro-ions under the influence of an electric field. A steady-state electrophoretic velocity, ve, attained by the migrating macro-ions is linearly proportional to the applied electric field. When a field is applied, the molecules' velocities are essentially always in equilibrium. To measure electrophoretic mobility, an electric field E is applied to drive electrophoresis of charged species, whose velocity ve is then measured to determine the electrophoretic mobility through the relationshipve=μE  (1)where μ is the electrophoretic mobility, or the velocity per unit electric field. An objective of the present invention is to provide an improved method for the measurement of the electrophoretic mobility of particles in solution.
Several techniques have been developed and are available for measuring electrophoretic mobility. Among these techniques are the moving boundary method, microelectrophoresis, and electrophoretic light scattering, ELS, which includes several light scattering methods including heterodyne dynamic light scattering, DLS, laser Doppler electrophoresis, LDE, and phase analysis light scattering, PALS. The electrophoretic mobility can also be measured by an electroacoustic means: electrokinetic sonic amplitude, ESA, as described by Oja, et.al. in U.S. Pat. No. 4,497,208, Issued Feb. 5, 1985, “Measurement of Electro-Kinetic Properties of a Solution.”
Free-solution measurements of electrophoretic mobility have been routinely carried out in the batch mode wherein a sample containing macromolecules of interest is loaded into an apparatus, an AC (alternating current) electric field is applied and the electrophoretic velocity is directly measured without externally imposed flow
In general, the movement of macromolecules can be diffusional, due to Brownian motion, and collective, due to electro-osmosis, electrophoresis, externally applied fluid flow, thermal convection, etc. In order to determine the electrophoretic component, the contributions to molecular motions from other mechanisms must be accounted for. The contribution from random Brownian motion is necessarily averaged out over multiple measurements. The effects of electro-osmosis can be ignored by measuring at the stationary layer, or by increasing the frequency of electric field reversal to suppress electro-osmosis. The contribution from thermal convection or residual bulk fluid flow can be subtracted out since it is independent of the direction of the applied electric field and shows up as a constant velocity component while the electrophoretic component switches polarity in synchronicity with the alternating electric field. Most instruments are able to account for thermal convection that is usually the undesirable byproduct of electrical currents. When it comes to the measurement of electrophoretic mobility, the conventional wisdom states that such measurements should be performed under minimum bulk fluid flow. A volume flow rate 0.2 mL/min or higher can easily overwhelm the mobility measurements.